Development and evaluation of pullulan-based composite antimicrobial films (CAF) incorporated with nisin, thymol and lauric arginate to reduce foodborne pathogens associated with muscle foods.

A novel composite antimicrobial film (CAF), made from a pullulan-based biopolymer and polyethylene (PE) was developed and evaluated for controlling pathogens associated with muscle foods. Initially, CAFs were developed by incorporating thymol (T), nisin (N) and/or lauric arginate (LAE) into the pullulan layer and layering it on top of PE. The antimicrobial activity of the resulting CAFs was evaluated against cocktails of Shiga toxin-producing E. coli (STEC), Salmonella spp., Listeria monocytogenes (L. monocytogenes) and Staphylococcus aureus (S. aureus) in disk diffusion assays (DDAs). CAFs containing N were ineffective, while those containing T were effective for inhibiting the pathogens in DDAs. However, CAFs made with them did not exhibit desirable physical and mechanical properties since solvents (HCl and ethanol, respectively) interfered with the binding of pullulan to PE. Conversely, CAFs made with 0.5, 1 and 2.5% LAE maintained proper physical and mechanical characteristics and inhibited the four bacterial pathogens in DDAs. Based on these preliminary results, cocktails consisting of approximately 8 log10 CFU/ml of STEC, Salmonella, L. monocytogenes, or S. aureus were experimentally-inoculated onto raw beef, raw chicken breast, or ready-to-eat (RTE) turkey breast to obtain approximately 6.6 log10 CFU/cm2, aseptically transferred to CAFs containing 0.5, 1, or 2.5% LAE that were made into sachets/bags, vacuum packaged, sealed, and remaining microbial populations determined up to 28 days of refrigerated storage (4 °C). By day 28, CAFs containing 0.5, 1, and 2.5% LAE reduced: STEC by 1.13, 1.33 and 2.88 log10 CFU/cm2 respectively, on raw beef; Salmonella by 2.03, 2.12 and 3.01 log10 CFU/cm2 respectively, on raw chicken breast; L. monocytogenes by 1.12, 1.81 and 3.56 log10 CFU/cm2 respectively, on RTE turkey breast; and S. aureus by 0.68, 2.02 and 3.43 log10 CFU/cm2, respectively, on RTE turkey breast. CAFs may be of interest to the meat and poultry industry to control foodborne pathogens associated with these food products.

Thermoplastic starch/polybutylene adipate terephthalate film coated with gelatin containing nisin Z and lauric arginate for control of foodborne pathogens associated with chilled and frozen seafood.

In order to control foodborne pathogens on seafood products, an antimicrobial, thermoplastic starch/polybutylene adipate terephthalate (TPS/PBAT; 40/60) film was produced by coating gelatin (15% v/v) containing lauric arginate (LAE; 0.8 mg/cm2), alone or combination with nisin Z (69.4 AU/cm2) to produce LAE-Gelatin-TPS/PBAT and Nisin-LAE-Gelatin-TPS/PBAT films, respectively. Both films were investigated for control of Vibrio parahaemolyticus ATCC 17802 and Salmonella Typhimurium ATCC 14028 on bigeye snapper (Lutjanus lineolatus) and tiger prawn (Penaeus monodon) slices during long-term (28 days), refrigerated (4 °C; chilled) and frozen (-20 °C) storage up to 90 days. S. Typhimurium ATCC 14028, experimentally inoculated onto bigeye snapper and tiger prawn slices, treated with the LAE-Gelatin-TPS/PBAT film, and stored at 4 °C was reduced 3.2 log10 CFU/g after 28 days and 7 log10 CFU/g after 21 days, respectively. Nisin-LAE-Gelatin-TPS/PBAT film reduced S. Typhimurium ATCC 14028 on bigeye snapper and tiger prawn slices 3.5 log10 CFU/g after 28 days and 7 log10 CFU/g after 14 days at 4 °C, respectively. The LAE-Gelatin-TPS/PBAT and Nisin-LAE-Gelatin-TPS/PBAT films and storage for 28 days at 4 °C reduced V. parahaemolyticus inoculated on chilled bigeye snapper slices approximately 2.6 and 4.2 log10 CFU/g, respectively. Both films reduced V. parahaemolyticus inoculated on chilled tiger prawn slices approximately 7.1 log10 CFU/g after 28 days at 4 °C. The LAE-Gelatin-TPS/PBAT and Nisin-LAE-Gelatin-TPS/PBAT films also reduced S. Typhimurium, inoculated on bigeye snapper and tiger prawn slices, 5.8 and 5.6 log10 CFU/g, respectively, after 60 days at -20 °C. V. parahaemolyticus was reduced by 5.8 log10 CFU/g on frozen bigeye snapper and tiger prawn slices after treatment with Nisin-LAE-Gelatin-TPS/PBAT film after 14 and 21 days, respectively. However, the LAE-Gelatin-TPS/PBAT film reduced V. parahaemolyticus 5.8 log10 CFU/g on both frozen seafood slices after 28 days. The results obtained from this study indicate the LAE-Gelatin-TPS/PBAT and Nisin-LAE-Gelatin-TPS/PBAT films displayed excellent inhibition against S. Typhimurium and V. parahaemolyticus on chilled and frozen seafood.

Prevalence and Phylogenetic Characterization of Escherichia coli and Hygiene Indicator Bacteria Isolated from Leafy Green Produce, Beef, and Pork Obtained from Farmers' Markets in Pennsylvania.

The popularity of farmers' markets in the United States has led to over 8,400 farmers' markets being in operation in 2015. As farmers' markets have increased in size and complexity in the kinds of foods sold at these venues, so have the potential food safety risks. Since 2008, seven major foodborne illness outbreaks and two recalls associated with food products from farmers' markets have occurred, causing 80 known reported illnesses and one death. Various researchers also have observed vendors performing high-risk food safety retail behaviors, and others have identified microbiological hazards in foods sold at farmers' markets. In this study, the presence of hygiene indicators (coliforms, fecal coliforms, Listeria spp., and Escherichia coli ) was assessed in select samples of leafy green produce and meat obtained from farmers' markets in Pennsylvania. E. coli isolates were further characterized by phylogenetic profile and virulence potential. E. coli was present in 40% (20 of 50) and 18% (9 of 50) of beef and pork samples, respectively, and in 28% (15 of 54), 29% (15 of 52), and 17% (8 of 46) of kale, lettuce, and spinach samples, respectively. Listeria spp. was found in 8% (4 of 50) of beef samples, 2% (1 of 54) of kale samples, 4% (2 of 52) of lettuce samples, and 7% (3 of 46) of spinach samples. Among the 10 Listeria spp. isolates, 3 were identified as L. monocytogenes . E. coli isolated from meat samples mainly clustered into phylogroup B1 (66%; 19 of 29), whereas produce isolates clustered into phylogroups B2 (36%; 14 of 39) and B1 (33%; 13 of 39). These E. coli isolates possessed the fimH, iroN, hlyD, and eae genes associated with extraintestinal pathogenic E. coli and Shiga toxin-producing E. coli . The high prevalence but low levels of E. coli and Listeria spp. found on both produce and meat products obtained from farmers' markets in this study strongly indicate that farmers' market vendors would benefit greatly from food safety training and increased public health oversight.

Commercially Available Rapid Methods for Detection of Selected Food-borne Pathogens.

Generally, the enumeration and isolation of food-borne pathogens is performed using culture-dependent methods. These methods are sensitive, inexpensive, and provide both qualitative and quantitative assessment of the microorganisms present in a sample, but these are time-consuming. For this reason, researchers are developing new techniques that allow detection of food pathogens in shorter period of time. This review identifies commercially available methods for rapid detection and quantification of Listeria monocytogenes, Salmonella spp., Staphylococcus aureus, and Shiga toxin-producing Escherichia coli in food samples. Three categories are discussed: immunologically based methods, nucleic acid-based assays, and biosensors. This review describes the basic mechanism and capabilities of each method, discusses the difficulties of choosing the most convenient method, and provides an overview of the future challenges for the technology for rapid detection of microorganisms.

Fate of Pathogenic Bacteria Associated with Production of Pickled Sausage by Using a Cold Fill Process.

Preservation by pickling has been used for many years to extend the shelf life of various types of food products. By storing meat products in a brine solution containing an organic acid, salt, spices, as well as other preservatives, the pH of the product is reduced, thus increasing the safety and shelf life of the product. Pickling may involve the use of heated brines to further add to the safety of the food product. When precooked, ready-to-eat (RTE) sausages are pickled with a heated brine solution, the process is referred to as hot filling. However, hot filling has been shown to affect the clarity of the brine, making the product cloudy and unappealing to consumers. Because of the potential quality defects caused by higher temperatures associated with hot fill pickling, cold fill pickling, which uses room temperature brine, is preferred by some pickled sausage manufacturers. Because little information exists on the safety of cold fill, pickled sausages, a challenge study was designed using a brine solution (5% acetic acid and 5% salt at 25°C) to pickle precooked, RTE sausages inoculated with a pathogen cocktail consisting of Salmonella Typhimurium, Salmonella Senftenberg, Salmonella Montevideo, Listeria monocytogenes , and Staphylococcus aureus . All pathogens were reduced ~6.80 log CFU/g in 72 h when enumerated on nonselective media. On selective media, Salmonella and L. monocytogenes decreased 6.33 and 6.35 log CFU/g in 12 h, respectively whereas S. aureus was reduced 6.80 log CFU/g in 24 h. Sausages experienced significant (P ≤ 0.05) decreases in pH over the 28 days of storage, whereas no significant differences were observed in water activity (P =0.1291) or salt concentration of the sausages (P =0.1445) or brine (P =0.3180). The results of this experiment demonstrate that cold fill pickling can effectively reduce and inhibit bacterial pathogens.

Incorporation of nisin Z and lauric arginate into pullulan films to inhibit foodborne pathogens associated with fresh and ready-to-eat muscle foods.

A combination of food grade compounds with edible films, used to inhibit foodborne pathogens associated with fresh or further processed muscle foods, is receiving considerable attention. In this study, pullulan films containing lauric arginate (LAE) and nisin Z (produced by Lactococcus lactis subsp. lactis I8-7-3 and isolated from catfish gut), alone or in combination, were investigated for controlling foodborne pathogens on fresh and further processed muscle foods after long-term refrigerated storage. Salmonella Typhimurium and Salmonella Enteritidis on raw turkey breast slices wrapped with a film containing LAE or the combination of LAE with nisin Z were reduced throughout the experiment, 2.5 to 4.5 log10 CFU/cm(2) and 3.5 to 5.1 log10 CFU/cm(2), respectively. Film containing a combination of LAE with nisin Z reduced Staphylococcus aureus and Listeria monocytogenes Scott A inoculated onto ham surfaces by approximately 5.53 and 5.62 log10 CFU/cm(2), respectively during refrigerated storage. Escherichia coli O157:H7, O111, and O26 also were reduced by >4 log 10CFU/cm(2) on raw beef slices after treatment with the combination film and refrigerated storage. The results obtained from this study indicate the LAE- and LAE-nisin Z-containing pullulan films displayed excellent inhibition against foodborne pathogens on fresh and further processed muscle foods.

Efficacy of antimicrobial pullulan-based coating to improve internal quality and shelf-life of chicken eggs during storage.

There has been a growing interest in the use of natural materials as a delivery mechanism for antimicrobials and coatings in foods. The aim of the present study was to evaluate the effectiveness of pullulan coatings to improve internal quality and shelf-life of fresh eggs during 10 wk of storage at 25 and 4 °C. Three treatments of eggs were evaluated as follows; non-coated (control; C), coated with pullulan (P), and coated with pullulan containing nisin (N). The effects of the pullulan coatings on microbiological qualities, physical properties, and freshness parameters were investigated and compared with non-coated eggs. For non-coated eggs, as storage time increased, yolk index, albumen index, and Haugh unit value decreased and weight loss increased. However, pullulan coatings (P or N) minimized weight loss (<1.5%) and preserved the albumen and yolk quality of eggs (with a final B grade) 3 wk longer than non-coated eggs at 25 °C. At 4 °C, both P- and N-coated eggs went from AA to A grade after 9 wk and maintained the grade for 10 wk (4 wk longer than that of non-coated eggs). This study is the first to demonstrate that pullulan coatings can preserve the internal quality, prolong the shelf-life, and minimize weight loss of fresh eggs.

Diversity of CRISPR loci and virulence genes in pathogenic Escherichia coli isolates from various sources.

Shiga toxin-producing Escherichia coli (STEC) strains, including those of O157:H7 and the "big six" serogroups (i.e., O26, O45, O103, O111, O121, and O145) are food-borne pathogens that pose a serious health threat to humans. Ruminants, especially cattle, are a major reservoir for O157 and non-O157 STEC. In the present study, 115 E. coli strains isolated from small and very small beef processing plants were screened for virulence genes (stx1, stx2, eae) using a multiplex polymerase chain reaction (PCR). Thirteen (11.3%) of the 115 isolates tested positive for stx1, stx2, or eae genes, but only 4 (3.5%) tested positive for either stx1 or stx2. A multiplex PCR reaction targeting eight O-serogroups (O26, O45, O103, O111, O113, O121, O145, O157) identified 12 isolates as O26, O103, O111, or O145, with E. coli O26 being the most predominant serogroup (61.5%). The thirteen isolates were further analyzed using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) subtyping. Consistent with previous studies, CRISPR alleles from strains of the same serogroup were similar in their spacer content and order, regardless of the isolation source. A completely different CRISPR allele was observed in one isolate ("7-J") which exhibited a different O-serogroup (O78). Our results confirmed previous findings that CRISPR loci are conserved among phylogenetically-related strains. In addition, 8 E. coli O26 isolates and a collection of 42 E. coli O26 isolates were screened for 12 enterohemorrhagic E. coli-specific genes. Seven genes (ECs848-Hypothetical Protein, ECs2226-Hypothetical Protein, ECs3857-nleB, ECs3858-Hypothetical Protein, ECs4552-escF, ECs4553-Hypothetical Protein, and ECs4557-sepL) were found in all 50 isolates. An additional 5 genes (ECs1322-ureA urease subunit γ, ECs1323-ureB urease subunit ß, ECs1326-ureF, ECs1561-Hypothetical Protein, and ECs1568-Hypothetical Protein) were found to be highly prevalent in isolates from human sources, while lower in isolates from beef processing plants, cattle, and other sources. This finding indicates the possible role of these genes in virulence of human O26 strains.

The efficacy of short and repeated high-pressure processing treatments on the reduction of non-O157:H7 Shiga-toxin producing Escherichia coli in ground beef patties.

High pressure processing (HPP) has previously been shown to be effective at reducing Escherichia coli O157:H7 in meat products. However, few studies have determined whether HPP may be effective at reducing non-O157:H7 Shiga toxin-producing E. coli (STEC) in ground beef. This study investigated the efficacy of short and repeated HPP treatments to reduce non-O157:H7 STEC inoculated into ground beef. Irradiated ground beef patties (80:20, 90:10 [lean:fat]) were inoculated with pairs of E. coli serogroups O103, O111, O26, O145, O121, O45, O157:H7, and DH5α, vacuum-packaged and high-pressure processed (four, 60 s cycles, 400 MPa, 17°C). Surviving E. coli populations were enumerated on Rainbow Agar O157 and Tryptic Soy Agar. HPP treatments produced >2.0 log10 CFU/g reductions of each E. coli serogroup, and reductions ranged from 2.35-3.88 and 2.26-4.31 log10 CFU/g in 80:20 and 90:10 samples, respectively. These results suggest that HPP could be an effective, post-processing intervention to reduce the risk of non-O157:H7 STEC contamination in ground beef.

Multivariate analysis reveals differences in biofilm formation capacity among Listeria monocytogenes lineages.

Biofilm formation capacity evaluated under identical conditions differs among Listeria monocytogenes lineages. The approach of using one set of factors or one variable at a time fails to explain why some lineages are more prevalent than others in certain environments. This study proposes the use of multivariate analysis to compare biofilm formation by various strains and describes the ecological niches of L. monocytogenes lineages. Nutrient availability, temperature, pH and water activity (aw) at three different levels were used to determine biofilm formation by 41 strains. Despite the high degree of similarity (≤ 80%), distinct lineage-associated biofilm formation patterns were identified. A linear regression model for each strain and a principal component analysis of regression coefficients indicated that Lineages I and III have different, but overlapping, ecological niches. This study is the first to report the use of multivariate analyses to compare biofilm formation by various isolates of L. monocytogenes.

Effect of lauric arginate, nisin Z, and a combination against several food-related bacteria.

The effects of lauric arginate (LAE) and nisin Z, alone or in combination, on cell damage were investigated against Escherichia coli O157:H7, Listeria monocytogenes and Brochothrix thermosphacta, by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations, efflux of potassium and phosphate ions, and growth inhibition. A combination of LAE with nisin Z caused severe and dramatic changes in the cytoplasmic membrane and cell lysis of both Gram-positive and Gram-negative bacteria. The combination treatment also caused significant potassium and phosphate ion leakage of E. coli O157:H7, L. monocytogenes and B. thermosphacta, when compared with other treatments: 16.62±1.05, 50.35±0.81 and 45.47±1.15mg/L of potassium ion and 122.66±8.81, 97.96±3.31 and 26.47±13.97mg/L of phosphate ion after treatment for 6h, respectively. Bacteria were reduced by approximately 7log10CFU/mL within the first hour of treatment and then cells were unable to grow for the remainder of the experiment. Treatment with LAE alone resulted in changes in cellular morphology, coagulation of the cytoplasm, and low level leakage of potassium and phosphate ions in all bacteria tested. Treatment of L. monocytogenes and B. thermosphacta with nisin Z (320AU/mL of final concentration) resulted in the formation of membrane channels and leakage of potassium and phosphate ions at rather high levels; but the bacteriocin was not effective against E. coli O157:H7. LAE or nisin Z reduced growth of both L. monocytogenes and B. thermosphacta by approximately 7log10CFU/mL. Conversely, E. coli O157:H7 was not inhibited by treatments with nisin Z, but decreased by approximately 4.45log10CFU/mL after treatment with LAE. These findings provide additional information on the mode of action of these compounds on bacterial populations.

Incorporation of essential oils and nanoparticles in pullulan films to control foodborne pathogens on meat and poultry products.

The incorporation of essential oils and nanotechnology into edible films has the potential to improve the microbiological safety of foods. The aim of this study was to evaluate the effectiveness of pullulan films containing essential oils and nanoparticles against 4 foodborne pathogens. Initial experiments using plate overlay assays demonstrated that 2% oregano essential oil was active against Staphylococcus aureus and Salmonella Typhimurium, whereas Listeria monocytogenes and Escherichia coli O157:H7 were not inhibited. Two percent rosemary essential oil was active against S. aureus, L. monocytogenes, E. coli O157:H7, and S. Typhimurium, when compared with 1%. Zinc oxide nanoparticles at 110 nm were active against S. aureus, L. monocytogenes, E. coli O157:H7, and S. Typhimurium, when compared with 100 or 130 nm. Conversely, 100 nm silver (Ag) nanoparticles were more active against S. aureus than L. monocytogenes. Using the results from these experiments, the compounds exhibiting the greatest activity were incorporated into pullulan films and found to inhibit all or some of the 4 pathogens in plate overlay assays. In challenge studies, pullulan films containing the compounds effectively inhibited the pathogens associated with vacuum packaged meat and poultry products stored at 4 °C for up to 3 wk, as compared to control films. Additionally, the structure and cross-section of the films were evaluated using electron microscopy. The results from this study demonstrate that edible films made from pullulan and incorporated with essential oils or nanoparticles may improve the safety of refrigerated, fresh or further processed meat and poultry products.

Inactivation of human pathogens during phase II composting of manure-based mushroom growth substrate.

Commercial production of white button mushrooms (Agaricus bisporus) requires a specialized growth substrate prepared from composted agricultural by-products. Because horse and poultry manures are widely used in substrate formulations, there is a need to determine the extent to which the composting process is capable of eliminating human pathogens. In this study, partially composted substrate was inoculated with a pathogen cocktail (log 106 to 108 CFU/g) containing Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella. Pathogen and indicator-organism reductions were followed at temperatures that typically occurred during a standard 6-day phase II pasteurization and conditioning procedure. Controlled-temperature water bath studies at 48.8, 54.4, and 60°C demonstrated complete destruction of the three pathogens after 36.0, 8.0, and 0.5 h, respectively. Destruction of L. monocytogenes and E. coli O157:H7 at 54.4°C occurred more slowly than E. coli, total coliforms, Enterobacteriaceae, and Salmonella. Microbial reductions that occurred during a standard 6-day phase II pasteurization and conditioning treatment were studied in a small-scale mushroom production research facility. After phase II composting, E. coli, coliforms, and Enterobacteriaceae were below detectable levels, and inoculated pathogens were not detected by direct plating or by enrichment. The results of this study show that a phase II composting process can be an effective control measure for eliminating risks associated with the use of composted animal manures during mushroom production. Growers are encouraged to validate and verify their own composting processes through periodic microbial testing for pathogens and to conduct studies to assure uniform distribution of substrate temperatures during phase II.

Presence of Shiga toxin-producing Escherichia coli O-groups in small and very-small beef-processing plants and resulting ground beef detected by a multiplex polymerase chain reaction assay.

Shiga toxin-producing Escherichia coli (STEC) are associated with foodborne illnesses, including hemolytic uremic syndrome in humans. Cattle and consequently, beef products are considered a major source of STEC. E. coli O157:H7 has been regulated as an adulterant in ground beef since 1996. The United States Department of Agriculture Food Safety and Inspection Service began regulating six additional STEC (O145, O121, O111, O103, O45, and O26) as adulterants in beef trim and raw ground beef in June 2012. Little is known about the presence of STEC in small and very-small beef-processing plants. Therefore, we propose to determine whether small and very-small beef-processing plants are a potential source of non-O157:H7 STEC. Environmental swabs, carcass swabs, hide swabs, and ground beef from eight small and very-small beef-processing plants were obtained from October 2010 to December 2011. A multiplex polymerase chain reaction assay was used to determine the presence of STEC O-groups: O157, O145, O121, O113, O111, O103, O45, and O26 in the samples. Results demonstrated that 56.6% (154/272) of the environmental samples, 35.0% (71/203) of the carcass samples, 85.2% (23/27) of the hide samples, and 17.0% (20/118) of the ground beef samples tested positive for one or more of the serogroups. However, only 7.4% (20/272) of the environmental samples, 4.4% (9/203) of the carcass samples, and 0% (0/118) ground beef samples tested positive for both the serogroup and Shiga toxin genes. Based on this survey, small and very-small beef processors may be a source of non-O157:H7 STEC. The information from this study may be of interest to regulatory officials, researchers, public health personnel, and the beef industry that are interested in the presence of these pathogens in the beef supply.

An ecological perspective of Listeria monocytogenes biofilms in food processing facilities.

Listeria monocytogenes can enter the food chain at virtually any point. However, food processing environments seem to be of particular importance. From an ecological point of view, food processing facilities are microbial habitats that are constantly disturbed by cleaning and sanitizing procedures. Although L. monocytogenes is considered ubiquitous in nature, it is important to recognize that not all L. monocytogenes strains appear to be equally distributed; the distribution of the organism seems to be related to certain habitats. Currently, no direct evidence exists that L. monocytogenes-associated biofilms have played a role in food contamination or foodborne outbreaks, likely because biofilm isolation and identification are not part of an outbreak investigation, or the definition of biofilm is unclear. Because L. monocytogenes is known to colonize surfaces, we suggest that contamination patterns may be studied in the context of how biofilm formation is influenced by the environment within food processing facilities. In this review, direct and indirect epidemiological and phenotypic evidence of lineage-related biofilm formation capacity to specific ecological niches will be discussed. A critical view on the development of the biofilm concept, focused on the practical implications, strengths, and weaknesses of the current definitions also is discussed. The idea that biofilm formation may be an alternative surrogate for microbial fitness is proposed. Furthermore, current research on the influence of environmental factors on biofilm formation is discussed.

Incidence of Shiga toxin-producing Escherichia coli strains in beef, pork, chicken, deer, boar, bison, and rabbit retail meat.

The objective of the current study was to determine the incidence of contamination by the top 7 Shiga toxin-producing Escherichia coli (STEC) O-groups, responsible for the majority of E. coli infections in human beings, in retail meat from different animal species. Samples from ground beef (n = 51), ground pork (n = 16), ground chicken (n = 16), and game meat (deer, wild boar, bison, and rabbit; n = 55) were collected from retail vendors for the detection of 7 STEC O-groups (O26, O45, O103, O111, O121, O145, and O157). Meat samples were tested by using a multiplex polymerase chain reaction assay targeting the wzx gene of O antigen gene clusters of the 7 STEC O-groups. The positive samples were further tested for Shiga toxin genes (stx1 and stx2). Out of a total of 83 ground beef, pork, and chicken samples, 17 (20%) carried O121, 9 (10%) carried O45, 8 (9%) carried O157, 3 (3%) carried O103, and 1 (1%) carried O145. None of the samples were positive for O26, O111, or the stx gene. All 3 white-tailed deer samples (100%) were positive for O45, O103, or both, 2 (10%) out of 20 red deer samples exhibited the presence of O103, and all 3 bison samples were contaminated with either O121, O145, or O157. One sample from ground deer, contaminated with E. coli O45, carried the stx1 gene. This preliminary investigation illustrates the importance of microbiological testing of pathogens in meat products, as well as the recognized need for increased surveillance and research on foodborne pathogens.

Investigation of chemical rinses suitable for very small meat plants to reduce pathogens on beef surfaces.

Numerous antimicrobial interventions are capable of reducing the prevalence of harmful bacteria on raw meat products. There is a need to identify effective and inexpensive antimicrobial interventions that could, in practice, be used in very small meat plants because of limited financial, space, and labor resources. Eight antimicrobial compounds (acetic acid, citric acid, lactic acid, peroxyacetic acid, acidified sodium chlorite, chlorine dioxide, sodium hypochlorite, and aqueous ozone) were applied at various concentrations with small, hand-held spraying equipment, and bactericidal effectiveness was examined. Beef plate pieces were inoculated with fecal slurry containing a pathogen cocktail (Escherichia coli O157:H7, Salmonella Typhimurium, Campylobacter coli, and Campylobacter jejuni) and natural populations of aerobic plate counts, coliforms, and E. coli. Antimicrobial solutions were applied to beef surfaces via a portable, pressurized hand-held spray tank, and treated surfaces were subjected to appropriate methods for the enumeration and isolation of pathogens and hygiene indicators. Relative antimicrobial effectiveness was determined (from greatest to least): (i) organic acids, (ii) peroxyacetic acid, (iii) chlorinated compounds, and (iv) aqueous ozone. Using the equipment described, a 2% lactic acid rinse provided 3.5- to 6.4-log CFU/cm(2) reductions across all bacterial populations studied. Conversely, aqueous ozone yielded 0.02- to 2.9-log CFU/cm(2) reductions in pathogens and hygiene indicators, and did not differ significantly from a control tap water rinse (P = 0.055 to 0.731). This 2% lactic acid rinse will be subsequently combined with a previously described water wash to create a multistep antimicrobial intervention that will be examined under laboratory conditions and validated in very small meat plants.

Detection of Shiga toxin-producing Escherichia coli O26, O45, O103, O111, O113, O121, O145, and O157 serogroups by multiplex polymerase chain reaction of the wzx gene of the O-antigen gene cluster.

O-antigens on the surface of Escherichia coli are important virulence factors that are targets of both the innate and adaptive immune system and play a major role in pathogenicity. O-antigens that are responsible for antigenic specificity of the strain determine the O-serogroup. E. coli O26, O45, O103, O111, O113, O121, O145, and O157 have been the most commonly identified O-serogroups associated with Shiga toxin-producing E. coli (STEC) implicated in outbreaks of human illness all over the world. A multiplex polymerase chain reaction assay was developed to simultaneously detect the eight STEC O-serogroups targeting the wzx (O-antigen-flippase) genes of all O-antigen gene clusters. The sensitivity of the multiplex polymerase chain reaction was found to be 10 colony forming units for each O-group when enriched in broth and 100 colony forming units when enriched in artificially inoculated apple juice diluted with tryptic soy broth for 16 h at 37°C. The method can be used for detecting STEC O-groups simultaneously and may be exploited for improving the safety of food products.

Multiplex PCR detection of Shiga toxin-producing Escherichia coli strains belonging to serogroups O157, O103, O91, O113, O145, O111, and O26 experimentally inoculated in beef carcass swabs, beef trim, and ground beef.

Numerous foodborne outbreaks are attributed to Shiga toxin-producing Escherichia coli (STEC) and have been recognized for causing gastrointestinal disease in humans. Beef products have been considered the principal source of STEC. A multiplex PCR assay enabling simultaneous detection of STEC O103, O91, O113, O145, O111, O157, and O26 was developed and evaluated in artificially contaminated beef carcass swabs, beef trim, and ground beef after overnight enrichment. Individual serogroups were experimentally inoculated at low (1 to 10 CFU/ml) and high (11 to 100 CFU/ml) levels, and with a cocktail of strains belonging to two, four, and six serogroups. There was no significant difference in detecting single STEC strains under the different conditions. Only when strains were combined were there significant differences in detection of all cocktail isolates in some of the beef products. To address this issue, four serogroups were experimentally inoculated together at three different estimated levels (10, 10(2), and 10(3) CFU/ml) in all three beef products. Results yielded no significant difference in detecting STEC at the three inoculation levels (10, 10(2), and 10(3) CFU/ml) in trim and carcass swabs, but there was a significant difference in detecting STEC at the lowest levels (10 and 10(2) CFU/ml) in the 80:20 nonirradiated ground beef, and in the detection of STEC in irradiated ground beef. The findings from this study could provide industry and government agencies with a tool to evaluate the prevalence and incidence of STEC in beef products and their processing environments.

Investigation of water washes suitable for very small meat plants to reduce pathogens on beef surfaces.

Water washing with a handheld hose was performed on beef surfaces to ascertain the most effective combination of methods needed to remove potentially harmful microorganisms. For these experiments, beef brisket surfaces were experimentally inoculated with a fecal slurry containing Escherichia coli O157:H7, Salmonella Typhimurium, Campylobacter coli, and Campylobacter jejuni. In a pilot study, surfaces were washed with cold water (15 degrees C) at various water pressures, spray distances, application times, and drip times, and remaining bacterial populations were determined following the enumeration and isolation of pathogens and naturally occurring hygiene indicators (mesophilic aerobic bacteria, coliforms, and E. coli). The most efficacious combinations of these washing conditions were applied subsequently to artificially contaminated beef brisket surfaces in conjunction with hot (77 degrees C), warm (54 degrees C), and additional cold (15 degrees C) water washes. In the cold water washing pilot study, combinations of physical washing conditions significantly reduced all bacterial populations (P < 0.05). Further studies clearly indicated the superior bactericidal effectiveness of hot water washing; E. coli O157:H7 and Salmonella Typhimurium were reduced by 3.8 and 4.1 log CFU/cm(2), respectively. Overall, higher water temperature, longer application times, and shorter spray distances more effectively removed pathogens from inoculated beef surfaces. These findings will be used to formulate water washing recommendations for very small meat processing establishments.